The genetic analysis of reproductive isolation between species of Drosophila has now reached the resolution necessary to start answering one of the fundamental questions of evolution: what is the genetic basis of species differences? A.H. Sturtevant, one of the founders of Drosophila genetics, was fascinated by this question and thought he had found a way to analyse it when he realized that 'Drosophila melanogaster' was actually two species: D. melanogaster and D. simulans. By passing genes between these two species he hoped to investigate their genetic differences directly. No doubt he was disappointed to find that the D. melanogaster/D. simulans hybridization resulted only in unisexual sterile hybrids, a disappointment appreciated all the more by modern evolutionary biologists. Seventy-five years after Sturtevant's description of D. melanogaster/D. simulans hybrid sterility, we have discovered a strain of D. simulans that produces fertile female hybrids in crosses with D. melanogaster. Our discovery promises to bring the enormous resolution of D. melanogaster genetics to the study of reproductive isolation and species differences.

In the last decade, the genetic basis of reproductive isolation has been shown to be surprisingly polygenic, and yet even the most efficient system currently in use could lend itself to molecular analysis only in highly selected cases. By extending the recent discovery of fertility rescue between Drosophila melanogaster and Drosophila simulans, we show that this hybridization can permit systematic and precise delineation of the genetic and molecular basis of speciation. In a region of 5% of the D. simulans genome introgressed into D. melanogaster, we discover at least six genes of hybrid male sterility and none for female sterility by deficiency mapping. A single case of hybrid inviability has been tracked down to a 3-Kb element that was inserted into the Cyclin E locus during species hybridization. The extent of interspecific genetic divergence underlying hybrid male sterility, especially in contrast with the low degree of inviability and female sterility, is far greater than expected from previous studies.O ne of the important subjects in speciation study is the genetic mechanism of reproductive isolation (1, 2). Drosophila melanogaster, with its wealth of resources, could allow the delineation of genes of reproductive isolation by genetic, cytological, and molecular means. Unfortunately, this species does not produce progeny beyond the F1 generation when crossed to any of its known sibling species, including Drosophila simulans (3-5). Since the time A. H. Sturtevant observed this sterility 80 years ago (3), evolutionary geneticists have relied on other species pairs. Th. Dobzhansky (6) was the first to do so, and many have since followed. Nevertheless, the progress has been slow (7), and not until recently has a gene of reproductive isolation been cloned from Drosophila (8). To move the field at a faster pace, a more efficient system involving D. melanogaster is highly desirable.Various attempts at bringing this species into speciation research have been made, including mating triploid D. melanogaster females to heavily irradiated D. simulans males to obtain the equivalent of F2s (9, 10), searching for sibling species that might be crosshybridizable (4, 5), examining far-flung populations that might be reproductively isolated (11, 12), and surveying for hybrid inviability rescue mutations (13-16). None has succeeded in making this ''model organism'' usable material for systematic studies of the genetics of speciation. The recent discovery of combinations of lines from D. melanogaster and D. simulans that yield fertile hybrid females has perhaps given us the best chance to tap into the resources of D. melanogaster (17). Materials and MethodsNomenclature. We shall briefly explain the conventional Drosophila nomenclature used in this study. For example, Df(2L) J39 (31C-D; 32D-E) denotes a deletion on the left arm of the second chromosome with the two breakpoints determined to be at the locations of 31C (or 31D) and 32D (or 32E) of the polytene chromosome, respectively. This deletion is named J39. Following this ...

Hybrid females from crosses between Drsophila melanogaster males and females of its sibling species, D. simulans, D. mauritiana, or D. sechellia die as embryos. This lethality is believed to be caused by incompatibility between the X chromosome of D. melanogaster and the maternal cytoplasm. Zygotic hybrid rescue (Zhr) prevents this embryonic lethality and has been cytogenetically mapped to a proximal region of the X chromosome of D. melanogaster, probably in the centromeric heterochromatin. We have carried out high resolution cytological mapping of Zhr using deficiencies and duplications of the X heterochromatin. Deletions of the Zhr+ gene from the hybrid genome exhibit the Zhr phenotype. On the contrary, addition of the wild-type gene to the hybrid genome causes embryonic lethality, regardless of sex. The Zhr locus has been narrowed down to the region covered by Dp(1;f)1162 but not covered Dp(1;f)1205, a chromosome carrying a duplication of heterochromatin located slightly distal to the In(1)sc8 heterochromatic break-point.

We have been analyzing genes for reproductive isolation by replacing Drosophila melanogaster genes with homologs from Drosophila simulans by interspecific backcrossing. Among the introgressions established, we found that a segment of the left arm of chromosome 2, Int(2L)S, carried recessive genes for hybrid sterility and inviability. That nuclear pore protein 160 (Nup160) in the introgression region is involved in hybrid inviability, as suggested by others, was confirmed by the present analysis. Male hybrids carrying an X chromosome of D. melanogaster were not rescued by the Lethal hybrid rescue (Lhr) mutation when the D. simulans Nup160 allele was made homozygous or hemizygous. Furthermore, we uniquely found that Nup160 is also responsible for hybrid sterility. Females were sterile when D. simulans Nup160 was made homozygous or hemizygous in the D. melanogaster genetic background. Genetic analyses indicated that the D. simulans Nup160 introgression into D. melanogaster was sufficient to cause female sterility but that other autosomal genes of D. simulans were also necessary to cause lethality. The involvement of Nup160 in hybrid inviability and female sterility was confirmed by transgene experiment.